Interpretive Summary: Prexisting variation within the wheat streak mosaic virus (WSMV) Type and Sidney 81 strains was biologically separated by limiting dilution inoculation. Genetic differences among limiting dilution subisolates (LDSIs) were detected by a single-strand conformation polymorphism assay and verified by nucleotide sequencing. A Sidney 81 LDSI was subjected to serial passage in wheat, corn, and barley. Genetic changes in consensus sequence for each viral lineage passaged were monitored by SSCP and nucleotide sequencing. After nine passages, the consensus sequence of each viral lineage differed by 1 to 5 nucleotides from the original Sidney 81 sequence. An identical nucleotide change occurred in 8 of 9 lineages and was most easily explained as a reversion event driven by selection. All other nucleotide substitutions accrued during passage appeared to be due to genetic drift. These results indicate that WSMV populations may change over time and that genetic drift may account for much of the divergence among the numerous, but closely related, WSMV genotypes that constitute complex field populations. Non-consensus sequence variation measured before and after serial passage remained constant in frequency, regardless of host species examined. New mutations generated during viral replication within a cell appear to be generated at a constant rate, however, most are sequestered in virions and and are not subject to further amplification. Thus, the WSMV population within a plant consists of two classes of mutants: (1) new mutants that have not been replicated and therefore not tested for relative fitness; and (2) a much smaller pool of older mutants that have been replicated and and subjected to the evolutionary processes of selection and genetic drift.

Technical Abstract:
Variation within the Type and Sidney 81 strains of wheat streak mosaic virus (WSMV) was assessed by single-strand conformation polymorphism (SSCP) analysis and confirmed by nucleotide sequencing. Limiting dilution subisolates (LDSIs) of each strain were evaluated for polymorphism in the P1, P3, NIa, and CP cistrons. Different SSCP patterns among LDSIs of a strain were associated with single nucleotide substitutions. Sidney 81 LDSI-S10 was used as founding inoculum to establish three lineages each in wheat, corn, and barley. The P1, HC-Pro, P3, CI, NIa, NIb, and CP cistrons of each lineage at passages 1, 3, 6, and 9 were evaluated for polymorphism. By passage 9, each lineage differed in consensus sequence from LDSI-S10. Most substitutions occurred within NIa and CP, although at least one change occurred in each cistron except HC-Pro and P3. Most consensus sequence changes among lineages- were independent, with substitutions accumulating over time. However, LDSI-S10 bore a variant nucleotide (G-6016) in NIa that was restored to A-6016 in eight of nine lineages by passage 6. This near-global reversion is most easily explained by selection. Examination of non-consensus variation revealed a pool of unique substitutions (singletons) that remained constant in frequency during passage in all host species examined. These results suggest that mutations arising by viral polymerase error are generated at a constant rate, but that most newly generated mutants are sequestered in virions and do not serve as replication templates. Thus, a substantial fraction of variation generated is static and yet to be tested for relative fitness. In contrast, non- singleton variation increased upon passage, suggesting that some mutants do serve as replication templates and may become established in a population.